Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions which block the continued progression of the replication fork. Although structural studies with the various yeast and human TLS Pols have indicated that they could function in TLS in highly specialized ways, the information available for their biological roles has been relatively meager; in particular, the biological role of Pol? has remained the least understood. In the proposed studies, we will use a combined genetic, biochemical, and structural approach to test the hypothesis that Pol? makes an important contribution to promoting replication through DNA lesions which impair Watson-Crick (W-C) base pairing or which protrude into the DNA minor groove. Furthermore, and most importantly, studies will be done to test the hypothesis that Pol? functions opposite DNA lesions in a much more error-free manner than opposite undamaged residues. In Aim 1, we will examine the roles of Pol? and other TLS Pols in human cells in promoting replication through N1-methyl adenine (1-MeA), which impairs W-C base pairing; a deaza derivative of N3-methyl adenine (3-dMeA), which protrudes into the DNA minor groove; and an N2-dG adduct of 1,3-butadiene (N2-dG, R-butadiene monoepoxide), which like 3-dMeA, is a minor groove lesion but chemically more complex. 1-MeA and 3-dMeA are generated from exposure to environmental alkylating agents and from endogenous cellular reactions, and 1,3-butadiene is an important industrial chemical and an environmental pollutant. For TLS analysis in human cells, we will utilize two different duplex plasmid systems, an SV40 origin-based plasmid and an EBV origin-based plasmid, and the relative contributions of Pol? and of other Pols to lesion bypass and to mutagenicity will be determined. In Aim 2, biochemical studies will be done to examine the proficiency of Pol? and of other Pols in synthesizing DNA opposite the 1-MeA, 3-dMeA, and N2-dG R-butadiene monoepoxide adducts. By steady-state kinetic analyses, we will determine the catalytic efficiency and fidelity of Pol? and of other Pols for inserting a nucleotide (nt) opposite each of these lesions and for extending from the inserted nt. In Aim 3, structures of Pol? in ternary complex with the 1-MeA, 3-dMeA, N2-dG R-butadiene monoepoxide, and also a (6-4) TT photoproduct will be determined to uncover the bases of Pol? ability to function opposite these DNA lesions in a predominantly error-free manner. Biochemical studies, and TLS studies in human cells, will be carried out to examine the effects of mutations in residues that help stabilize the correct incoming nt opposite the lesion site. The proposed studies are highly relevant for cancer biology and cancer etiology as they will reveal whether Pol?, in conjunction with other TLS Pols, promotes a predominantly error-free mode of TLS opposite a diverse array of DNA adducts. An error-free mode of TLS would be in keeping with a role for Pol? in suppression of carcinogenesis that would otherwise result from exposure to environmental and chemical carcinogens.